Dissecting Cooperative and Additive Binding Energetics in the Affinity Maturation Pathway of a Protein-Protein Interface

Jianying Yang, Chittoor P. Swaminathan, Yuping Huang, Rongjin Guan, Sangwoo Cho, Michele C. Kieke, David M. Kranz, Roy A. Mariuzza, Eric J. Sundberg

Research output: Contribution to journalArticlepeer-review


When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vβ domain that exhibit an overall affinity range of ∼1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vβ domain, exhibit negative cooperativity.

Original languageEnglish (US)
Pages (from-to)50412-50421
Number of pages10
JournalJournal of Biological Chemistry
Issue number50
StatePublished - Dec 12 2003

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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